ABSTRACT
Two-photon microscopy is a powerful method in biomedical research that allows functional and anatomical imaging at a subcellular resolution in vivo. The technique is seriously hampered by absorption and scattering of light by blood, which prevents imaging through large vessels. Here, we demonstrate in the rat cerebral cortex that blood replacement by perfluorocarbon emulsion, a compound also used in human critical care medicine, yields superior image quality, while preserving neuronal integrity. Shadows of large superficial vessels disappear completely and cells can be imaged underneath them. For the first time, it is possible to image complete populations of neurons and astrocytes in the upper layers of neocortex in vivo.
Subject(s)
Blood Substitutes , Fluorocarbons , Microscopy, Confocal/methods , Microscopy, Fluorescence, Multiphoton/methods , Animals , Astrocytes/cytology , Astrocytes/metabolism , Blood Transfusion , Calcium Signaling , Humans , Male , Neurons/cytology , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Somatosensory Cortex/blood supply , Somatosensory Cortex/cytology , Somatosensory Cortex/metabolismABSTRACT
Ultrastructural features and morphometric values of ovine Sertoli and spermatogenic cells are reported with special reference to the 6 stages of the seminiferous epithelial cycle. Seminiferous tubules occupy about 83% of the testicular parenchyma. Average tubular diameter (about 275 microns) and epithelial height (about 95 microns) do not vary significantly during the cycle. From preleptotene to late diplotene the cellular volume of primary spermatocytes increases nearly five-fold; the nuclear volume increases three-fold in the same period. Secondary spermatocytes are observed exclusively during stage 4 of the seminiferous epithelial cycle. Due to partial cell necrosis and autolytic events, ovine spermatids lose a considerable amount of their cytoplasm during acrosome and maturation phases prior to spermiation. Sertoli cells occupy between 27.6% (stage 3) and 36.6% (stage 1) of the tubular epithelium. The average volume of a Sertoli cell varies between 6380 microns 3 (stage 2) and 7195 microns 3 (stage 4), the absolute surface area between 10550 microns 2 and 12305 microns 2. The irregularly contoured Sertoli cell nucleus contains a vesicular nucleolus and occupies about 7.5% of the cell. Mitochondria (about 5%) and smooth endoplasmic reticulum are other prominent organelles, but three-quarters of the Sertoli cell are taken up by cytoplasmic matrix with a well-developed cytoskeleton. Ovine Sertoli cells contain large basal lipid droplets, but no typical lipid cycle can be observed.
